In the previous years we have assigned four core domains of cytochrome c and their folding order. The previous studies have also suggested that the folding of cytochrome c may be driven by the core domain-domain interaction. Since 1988 several groups of other investigators have identified folding intermediates of different proteins which appear to be consistent with our concept of the core domains. Thus, the core domain-domain interaction may be general in driving the folding of globular proteins. To know whether or not the core domain-domain interaction differs from the known types of non-covalent interactions we have determined the differences in their thermodynamic and kinetic effects of substitution with norvaline (Nva) between Leu32 and Leu35 of the horse cytochrome c three-fragment complex in the previous years. The complex consists of a heme fragment of residues 1 to 5, (1-25)H, and two apofragments (28-38) and (39-104). It resembles native cytochrome c except for residues 39 to 55 which are flexible. We have now analyzed and interpreted the data using complex models. The results suggest for the first time that the difference in the standard Gibbs energy change, ddGs, associated with the equilibrium constant of fragment (28-38) with the ferric or ferrous complex qualitatively correlates with the difference in the activation Gibbs energy, dGa, associated with the direct dissociation of fragment (39-104). These ddGs and dGa also qualitatively correlate with the difference in the heat stability of the 695 nm band of the ferric complex, a band known to be indicative of the Fe-S bond. These correlations have been interpreted as suggesting that some propagative extra non-covalent interactions may be generated by the packed atomic groups in the hydrophobic core to stabilize the ground state. These interactions may have characteristics such that the stabilizing energy generated by them would be perturbed much more by the Leu32 Nva substitution than the Leu35 Nva. The perturbation of these interactions imposed by the removal of the gamma-methyl group of Leu32 would propagate itself through the core and affect the stability of the Fe- S bond and the binding strength of (39-104). These properties are consistent with the core domain-domain interaction. Furthermore, the analysis, combined with other evidence, appears to suggest that these propagative non-covalent interactions in the core are likely to differ from van der Waals interactions, hydrophobic energy, hydrogen bonds and charge-charge interactions. To map the core side chains involved in these propagative non-covalent interactions studies of yeast iso-2-cytochrome c using site directed mutagenesis is in progress. To date eight mutants have been or are being generated. To precisely identify the core domains of cytochrome c NMR studies are also in progress using two-dimensional NOESY, COSY, and TOCSY spectra in collaboration with the structural Biology Section, LBC, NHLBI.